We have recently shown that oxytocin inhibits cell growth when the vast majority of oxytocin receptors (OTRs) are excluded from detergent-resistant membranes (DRMs, the biochemical counterpart of lipid rafts), but has a strong mitogenic effect when the receptors are targeted to these plasma membrane domains upon fusion with caveolin2, a resident raft protein. The aim of this study was to investigate whether the manipulation of total cell cholesterol can influence OTR localisation and signalling. Our data indicate that cholesterol depletion in HEK293 cells does not affect the signalling events mediated by the OTRs located outside DRMs: when treated with 2 mM methyl--cyclodextrin (MCD), the receptors remained outside and continued to inhibit cell growth. On the contrary, the MCD treatment of cells expressing receptors fused to caveolin-2 led to their redistribution outside DRMs, and converted the receptor-mediated proliferative effect into cell growth inhibition. These data indicate that: 1) once released from DRMs, the receptors fused to caveolin2 signal are exactly the same as wild-type OTRs; and 2) their DRM location is responsible for the specific OTR signalling leading to cell proliferation. Finally, we evaluated whether cholesterol loading could force the OTRs into lipid rafts and change their signalling but, after cell treatment with an MCD/cholesterol complex, receptor stimulation continued to lead to cell growth inhibition, thus indicating that increasing cell cholesterol levels is not sufficient per se to affect OTR signalling.